Nucleic acids, proteins and polysaccharides are three major classes of biopolymers. While the first two systems are principally linear assemblies, polysaccharides are structurally more complex. This structural and stereochemical diversity results in a rich content of xe2x80x9cinformationxe2x80x9d in relatively small molecules. Nature further xe2x80x9cleveragesxe2x80x9d the structural versatility of polysaccharides by their covalent attachment (i.e., xe2x80x9cconjugationxe2x80x9d) to other biomolecules such as isoprenoids, fatty acids, neutral lipids, peptides or proteins. Oligosaccharides in the form of glycoconjugates mediate a variety of events including inflammation, immunological response, metastasis and fertilization. Cell surface carbohydrates act as biological markers for various tumors and as binding sites for other substances including pathogens.
Moreover, many physiologically important recognition phenomena involving carbohydrates have been discovered in recent years. Lectins, proteins which contain carbohydrate recognition domains, have been identified. Prominent members of the calcium dependent (C-type) lectin family (Drickamer, K. Curr. Opin. Struct. Biol. 1993, 3, 393) are the selectins which play a crucial role in leukocyte recruitment in inflammation. Bevilacqua, M. P.; Nelson, R. M. J. Clin. Invest. 1993, 91, 379. Members of the C-type lectin superfamily have been described on NK cells and Ly-49, NKR-P1 and NKG2 constitute group V of C-type lectins. While many lectins have been purified and cloned, their ligands have not been identified due to the heterogeneous nature of carbohydrates.
The recognition that interactions between proteins and carbohydrates are involved in a wide array of biological recognition events, including fertilization, molecular targeting, intercellular recognition, and viral, bacterial and fungal pathogenesis, underscores the importance of carbohyrates in biological systems. It is now widely appreciated that the oligosaccharide portions of glycoproteins and glycolipids mediate certain recognition events between cells, between cells and ligands, between cells and the extracellular matrix, and between cells and pathogens. See, e.g., U.S. Pat. No. 4,916,219 (describing oligosaccharides with heparin-like anticomplement activity).
These recognition phenomena may be inhibited by oligosaccharides having the same sugar sequence and stereochemistry found on the active portion of a glycoprotein or glycolipid involved in the recognition phenomena. The oligosaccharides are believed to compete with the glycoproteins and glycolipids for binding sites on the relevant receptor(s). For example, the disaccharide galactosyl-xcex2-1-4-N-acetylglucosamine is believed to be one component of the glycoproteins which interact with receptors in the plasma membrane of liver cells. Thus, to the extent that they compete with moieties for cellular binding sites, oligosaccharides and other saccharide compositions have the potential to open new horizons in pharmacology, diagnosis, and therapeutics.
The growing appreciation of the key roles of oligosaccharides and glycoconjugates in fundamental life sustaining processes has stimulated a need for access to usable quantities of these materials. Glycoconjugates are difficult to isolate in homogeneous form from living cells since they exist as microheterogeneous mixtures. The purification of these compounds, when possible, is at best tedious and generally provides only very small amounts of the compounds. The travails associated with isolation of oligo- and poly-saccharides and glycoconjugates from natural sources present a major opportunity for the development and exploitation of chemical synthesis. See, e.g., U.S. Pat. Nos. 4,656,133; 5,308,460; 5,514,784; and 5,854,391 (describing representative means of glycosylating saccharides and peptides).
Intense work is ongoing on the further development of the use of biologically-active oligosaccharides within a number of different fields, including novel diagnostics and blood typing reagents; highly specific materials for affinity chromatography; cell specific agglutination reagents; targeting of drugs; monoclonal antibodies, e.g., against cancer-associated reagents; as an alternative to antibiotics, based on the inhibition with specific oligosaccharides of the attachment of bacteria and viruses to cell surfaces; and stimulation of the growth of plants and protection of them against pathogens. Additionally, a considerable future market is envisaged for fine chemicals based on biologically-active carbohydrates.
As stated above, due to the difficulties associated with purification of glycoconjugates and oligosaccharides from natural sources, chemical synthesis may be the only way to procure sufficient amounts of these structures for detailed biochemical and biophysical studies. Additionally, combinatorial carbohydrate libraries hold great potential for the identification of carbohydrate-based ligands to cellular receptors. Identification of these molecules will open many new avenues for the development of diagnostic tools and therapeutic agents.
The invention of solid phase peptide synthesis by Merrifield 35 years ago dramatically influenced the strategy for the synthesis of these biopolymers. The preparation of structurally defined oligopeptides (Atherton, E.; Sheppard, R. C. Solid phase peptide synthesis: A practical approach; IRL Press at Oxford University Press: Oxford, England, 1989, pp 203) and oligonucleotides (Caruthers, M. H. Science 1985, 230, 281) has benefited greatly from the feasibility of conducting their assembly on various polymer supports. The advantages of solid matrix-based synthesis, in terms of allowing for an excess of reagents to be used and in the facilitation of purification are now well appreciated. However, the level of complexity associated with the synthesis of an oligosaccharide on a polymer support dwarfs that associated with the other two classes of repeating biooligomers. First, the need to differentiate similar functional groups (hydroxyl or amino) in oligosaccharide construction is much greater than the corresponding needs in the synthesis of oligopeptides or oligonucleotides. Furthermore, in these latter two cases, there is no stereoselection associated with construction of the repeating amide or phosphate bonds. In contrast, each glycosidic bond to be fashioned in a growing oligosaccharide ensemble constitutes a new locus of stereogenicity.
Combinatorial chemistry has been used in the synthesis of large numbers of structurally distinct molecules in a time and resource-efficient manner. Peptide, oligonucleotide, and small molecule libraries have been prepared and screened against receptors or enzymes to identify high-affinity ligands or potent inhibitors. These combinatorial libraries have provided large numbers of compounds to be screened against many targets for biological activity. Every pharmaceutical company now devotes a major effort to the area of combinatorial chemistry in order to develop new lead compounds in a rapid fashion.
The development of protocols for the solid support synthesis of oligosaccharides and glycopeptides requires solutions to several problems. Of course, considerable thought must be addressed to the nature of the support material. The availability of methods for attachment of the carbohydrate from either its xe2x80x9creducingxe2x80x9d or xe2x80x9cnon-reducingxe2x80x9d end would be advantageous. Also, selection of a linker which is stable during the synthesis, but can be cleaved easily when appropriate, is critical. A protecting group strategy that allows for high flexibility is desirable. Most important is the matter of stereospecific and high yielding coupling reactions.
Combinatorial carbohydrate libraries hold a tremendous potential with regard to therapeutic applications. The key role complex oligosaccharides play in biological processes such as inflammation, immune response, cancer and fertilization makes them highly attractive therapeutic targets. The ability to create true oligosaccharide libraries has the potential to trigger a revolution in the area of biopharmaceuticals.
The generation of combinatorial carbohydrate libraries will facilitate the rapid identification of ligands to many carbohydrate binding proteins which are involved in a variety of important biological events including inflammation (Giannis, A. Angew. Chem. Int. Ed. Engl. 1994, 33, 178), immune response (Ryan, C. A. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 1) and metastasis (Feizi, T. Curr. Opin. Struct. Biol. 1993, 3, 701). Analogs of ligands can help to define important lectin-ligand interactions. Non-natural ligands can be powerful inhibitors of carbohydrate-protein binding and will facilitate the study of cascade-like events involving such interactions. Furthermore, inhibitors of carbohydrate-lectin binding are potential candidates for a variety of therapeutic applications.
Moreover, the development of an automated oligosaccharide synthesizer holds great potential to influence glycobiology just as the peptide synthesizer impacted protein research. A reliable strategy for the solid support synthesis of oligosaccharides depends to a great extent on the choice of the linker which is used to anchor the first building block to the polymeric matrix.
A set of versatile linkers is described which are stable to a wide range of reaction conditions but can be cleaved in several ways to produce free oligosaccharides, fully-protected oligosaccharide building blocks and novel glycoconjugates. Furthermore, these linkers may be used to attach to solid supports building blocks useful in the assembly of libraries of other types of small molecules.
In certain embodiments, the present invention relates to versatile linkers for tethering a molecule to a solid support, e.g., for tethering a monomer, oligomer or polymer to a solid support, which are stable to a wide range of reaction conditions, but can be cleaved under well-defined conditions, thereby liberating said molecule from the solid support. In preferred embodiments, the linkers of the present invention are used to tether to the solid support unprotected, partially-protected or fully-protected monosaccharides or oligosaccharides, or unprotected, partially-protected or fully-protected glycoconjugates. In other embodiments, the linkers of the present invention may be used to tether to solid supports building blocks useful in the assembly of libraries of other types of small molecules. In certain embodiments, the present invention relates to a molecule or plurality of molecules tethered to the solid support via a linker or linkers of the present invention.
In certain embodiments, the present invention relates to processes for synthesizing molecules, e.g., monomers, oligomers or polymers, on a solid support, wherein a starting material in the synthesis of said molecule, intermediates in the synthesis of said molecule, and said molecule itself are tethered to the solid support during the process via one of the linkers of the present invention. In certain processes of the present invention, the molecule is liberated from the solid support by cleavage of the linker of the present invention.
The invention described herein is expected to enable the automated synthesis of oligosaccharides and glycoconjugates in much the same fashion that peptides and oligonucleotides are currently assembled. The ability to synthesize defined biologically important glycoconjugates will be far reaching with many direct applications to biomedical questions. Opportunities for the application of the present invention include the development of automated oligosaccharide synthesis machines.